X-ray laser microscopy sample analysis system and method

ABSTRACT

Improved system and method of X-ray laser microscopy that combines information obtained from both X-ray diffraction and X-ray imaging methods. At least one sample is placed in an ultra-cold, ultra-low pressure vacuum chamber, often using a sample administration device configured to present a plurality of samples. The sample is exposed to brief bursts of coherent X-ray illumination, often further concentrated using X-ray mirrors and pinhole collimation methods. Higher resolution data from the samples is obtained using hard X-ray lasers, such as free electron X-ray lasers, and X-ray diffraction methods. Lower resolution data from the same samples can be obtained using any of hard or soft X-ray laser sources, and X-ray imaging methods employing nanoscale etched zone plate technology. In some embodiments both diffraction and imaging data can be obtained simultaneously. Data from both sources are combined to create a more complete representation of the samples.

CROSS REFERENCE TO RELATED APPLICATIONS

This invention is a continuation in part of U.S. patent application Ser.No. 15/218,017 “IMPROVED X-RAY LASER MICROSCOPY SYSTEM AND METHOD”,filed Jul. 23, 2016, now U.S. Pat. No. 9,583,229, issued Feb. 28, 2017;the entire contents of this application are incorporated herein byreference.

BACKGROUND OF THE INVENTION

Field of the Invention

This invention is in the field of X-ray laser microscopy.

Description of the Related Art

The resolving power of conventional optical microscopes is limited bythe wavelength of light. Thus over the years, various methods employingshorter wavelength particles, such as electrons, and shorter wavelengthphotons, such as X-rays, have been employed to achieve still higherlevels of resolution.

X-ray radiation spans a range of wavelengths in the nanometer region,generally ranging from about 10 nanometers (soft X-rays) with energiesof around 100 electron volts, to about 0.01 nanometers (hard X-rays)with energies up to about 10,000 electron volts. Photons with energiesabove this are typically referred to as gamma rays. Since many molecularbiological structures of interest have structural details in roughly the10 to 0.1 nanometer range, scientists have turned to X-rays to helpelucidate the structure of proteins, DNA, membranes, viruses, cells, andother biological structures of interest.

Due to the desirable aspects of coherent light sources, X-ray lasers areparticularly useful for X-ray microscopy. Here various methods have beenemployed, including X-ray diffraction methods, X-ray holographicmethods, and various types of X-ray imaging methods. Although X-raymirrors and lenses are much more difficult to fabricate than theiroptical counterparts, various methods of focusing X-rays, includingWolter, Kirkpatrick-Baez, and Schwarzschild x-ray mirror designs, aswell as various types of zone plate methods (which operate according todiffraction methods) have been devised.

Some recent improvements in zone plate technology include the work ofChang and Sakdinawat, “Ultra-high aspect ratio high-resolutionnanofabrication for hard X-ray diffractive optics” NATURECOMMUNICATIONS, 5:4243, Jun. 27, 2014, pages 1-7 (DOI:10.1038/ncomms5243). They taught that improved zone plates can beproduced using some of the same photolithographic methods used toproduce modern integrated circuits and other nanostructures. Thesemethods are also described by their US patent application 2015/037679.

Previous work in the X-ray microscopy field includes the soft X-rayimaging methods of Suckwer et. al., U.S. Pat. No. 5,177,774; Chao et.al, “Soft X-ray microscopy at a spatial resolution better than 15 nm”,NATURE, Vol 435, Jun. 30, 2005, pages 1210 to 1213; and Kirtz et. al.,“Soft X-ray microscopes and their biological applications”, Q. Rev.Biophys. 28, 33-130 (1995).

Other workers have used ultra-short bursts of hard X-rays, oftenproduced by free electron lasers, and X-ray diffraction methods, toelucidate structures at still higher resolution. This work includesGaffney et. al., “Imaging Atomic Structure and Dynamics with UltrafastX-ray Scattering”, Science 316, Jun. 8, 2007, pages 1444-1448.

Despite these advances, the field remains challenging. The highestresolution laser microscopy sample details can generally only beobtained by using ultra-short hard X-rays, which are difficult to focus,and which tend to rapidly destroy the sample. Here, use of X-raysproduced by free electron lasers, ultra-short bursts of energy, andX-ray diffraction methods (which do not require the sample scatteredX-rays to be imaged) are favored.

By contrast, lower resolution laser microscopy, using soft X-rays, is abit closer to conventional microscopy. Coherent soft X-ray light can beproduced by a variety of different methods, and the lower energies tendto be less destructive of the sample. The soft X-rays, after beingscattered by the sample, can be focused by various methods, such asvarious phase zone plate methods, resulting in a more conventional typemagnified image of the sample.

Another problem is that the performance of existing X-ray lasermicroscopes can be limited by other effects, such as X-ray scattering orattenuation off of residual air molecules in the vacuum chamber, and bythermal effects (e.g. heating of the sample and X-ray lenses).

Another problem is sample administration. Although some prior artmethods, such as the methods of Gaffney, are configured to analyze morethan one sample by sending particle stream composed of a plurality ofsingle particles through the X-ray beam, such methods can havedrawbacks.

BRIEF SUMMARY OF THE INVENTION

The invention is based, in part, on the insight that the lowerresolution sample structural information produced by prior art hardX-ray laser systems, which rely on X-ray laser diffraction methods, isfrequently lost. This is because larger (e.g. lower resolution) samplestructural details do not end up substantially diffracting the angle ofthe X-ray laser beam. Instead, larger sized structural details onlycause the X-rays to be diffracted at low angles from the main X-raybeam. Thus these larger sample structural details are not well separatedfrom X-rays that pass completely through the sample (116) to anysignificant extent. This often shows up as the central “sun” in a“sunburst” type X-ray diffraction pattern. As an example, see Gaffneyand Chapman, Science 316, 1444 (2007), FIG. 3A and 3B, which illustratehow the center region of an X-ray diffraction pattern is essentiallyunreadable.

This invention is also based, in part, on the insight that this missinglow resolution structural information could be provided by use ofalternative X-ray imaging methods, such as phase zone plate methods, andthat if the phase zone plate was positioned in an X-ray diffractionapparatus so that the phase zone plate only obscured a small portion ofthe center of the X-ray diffraction detector, little X-ray diffractioninformation would be lost because, as discussed above, the centralportion of an X-ray diffraction detector has little value due tointerference from the main X-ray beam that has passed completely throughthe sample.

The invention is also based, in part, on the insight that in thisregard, advances in high-resolution nanofabrication methods have allowedfor the fabrication of ultra-small zone plates capable of focusing evenhard X-rays, as well as soft X-rays. Collimator devices with ultra-small(nanometer scale) collimator openings may also be produced using thesemethods.

The invention is also based, in part, on the insight that for higherperformance, every effort must be made to reduce the damaging effects ofX-ray beams on both the sample and on the various X-ray microscopecomponents. In this regards, greater efforts to chill the devices toultra-cold temperatures, by use of cryogenic fluids, possibly includingeven liquid helium, would be desirable. Further, to improve contrast,every effort must also be made to reduce the vacuum of the microscopychamber to unusually low pressure levels.

The invention is also based, in part, on the insight that improvedmethods of sample handing and administration would also be useful.Ideally the system should be able to handle multiple types of samples ina single run. Thus, as will be discussed, in some embodiments, themethod may also use a sample dispensing device, such as an automateddispensing device, configured to present a plurality of samples, whichmay be different types of samples, and analyze this plurality ofdifferent samples during a single observing session. Such X-raymicroscopy facilities are typically quite expensive, and use of improveddispensing devices and methods, configured for more optimal methods ofsample delivery, can allow for more rapid data collection during a givenmicroscopy session. This in turn makes such X-ray microscopy facilitiesmore feasible for high volume use, and can open up the use of facilitiesto handle a greater variety of research problems in a more costeffective manner.

Thus in some embodiments, the invention may be an improved X-ray lasermicroscopy method, equipped to analyze a given sample (and often aplurality of given samples) using (sometimes simultaneously) both X-raydiffraction and X-ray imaging methods onto different X-ray detectorregions. Typically the higher resolution sample data will be obtainedfrom the X-ray diffraction portion of the device, while the lowerresolution sample data will be obtained from the X-ray imaging portionof the device. Both the X-ray diffraction data and the X-ray imagingdata can be fed to a computer, which can be configured to use both setsof information to produce a combined view of the sample that providesmore structural information than either one method could produce byitself.

To obtain X-ray imaging data with minimum loss of X-ray diffractiondata, and with as short a wavelength of X-ray radiation as possible, thesystem may often make use of nanoscale etched zone plate technology.

The efficiency of the improved X-ray microscope can further be enhancedby chilling various portions of the device to ultra-low temperatures,and maintaining ultra-low vacuum during operation.

This method will typically use at least one X-ray laser providingcoherent X-rays through an X-ray port (or other opening) into a vacuumchamber. Inside the vacuum chamber, a first X-ray optical device, suchas an X-ray mirror device, will focus or collimate the coherent X-raysonto the sample.

Improved sample handling techniques, to enable higher sample throughput,will also be taught.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an overview of the improved X-ray laser microscopy method.

FIG. 2A shows a detail of some of the various X-ray laser microscopycomponents previously shown in FIG. 1.

FIG. 2B, shows a drawing of the first outer portion of the X-raydetection apparatus as seen from an orthogonal angle.

FIG. 2C shows a drawing of the second X-ray optical device (e.g. phasezone plate) as seen from an orthogonal angle.

FIG. 2D shows a drawing of the second inner portion of the X-raydetection apparatus as seen from an orthogonal angle

FIG. 3A again shows a detail of some of the various X-ray lasermicroscopy components previously shown in FIG. 1 and FIG. 2A.

FIG. 3B shows that in some embodiments, the first X-ray optical device(e.g. the X-ray mirror device) may be cooled by running a lowtemperature fluid, such as liquid helium, through various hollowchannels.

FIG. 3C shows that in some embodiments, the second X-ray optical device(e.g. the X-ray phase zone plate) may also be cooled by running a lowtemperature fluid, such as liquid helium, through various hollowchannels.

FIG. 4 shows some additional components, such as a vacuum chamber, thatare also used in the improved X-ray laser microscopy method.

FIG. 5A shows an alternative embodiment where the second X-ray opticaldevice (phase zone plate) and the second inner portion of the detectionapparatus are disposed in a first alternative configuration

FIG. 5B shows a different alternative embodiment where the second X-rayoptical device (phase zone plate) and the second inner portion of theoptical detection apparatus are disposed in a second alternativeconfiguration.

FIG. 6 shows a first alternative method of sample handling.

FIG. 7 shows a second alternative method of sample handling.

FIG. 8 shows a third alternative method of sample handling.

FIG. 9A shows a front view of a device useful for a fourth alternativemethod of sample handling.

FIG. 9B shows a side view of the device from FIG. 9A.

FIG. 9C shows the device from FIG. 9A and 9B in the context of themicroscope's vacuum chamber.

FIG. 10 shows a fifth alternative method of sample handling.

DETAILED DESCRIPTION OF THE INVENTION

Here it will be useful to refer to both FIG. 1 and FIG. 4.

FIG. 1 shows an overview of the improved X-ray laser microscopy method,while FIG. 4 gives more details about the microscope's vacuum chamber,vacuum pump system, and X-ray port arrangement.

In some embodiments, the invention may be a system, device or method ofX-ray laser microscopy, typically using at least one X-ray laser (notshown), which will supply a coherent source of X-ray illumination (100),typically through an X-ray port (402) into a vacuum chamber (400)configured to hold many of the other X-ray microscope components. In thefollowing description, unless otherwise specified or drawn, assume thatthe various methods and components that are described are inside of avacuum chamber (400).

Note that typically the X-ray port will be composed of a substantiallyvacuum tight but X-ray resistant material. However in the case where theX-ray laser itself is under the same vacuum conditions as the vacuumchamber, then the X-ray port may be a simple opening in the side of thevacuum chamber.

This coherent source of X-ray illumination can be provided by varioustypes of X-ray lasers, and indeed in some embodiments, it may be usefulto use more than one type of X-ray laser (e.g. a hard X-ray laser and asoft X-ray laser). These X-ray lasers can be any of a free-electronlaser, capillary plasma-discharge laser, solid-slab target laser,optical field excited plasma laser, high-harmonic generation laser, andthe like.

Since the beam size of coherent X-ray light sources (100) is usuallysignificantly larger than the size of the microscopy sample (108), itwill typically be useful to use a first X-ray optical device (102)configured to focus and/or collimate the X-rays onto the microscopysample (sample) (108). A sample administrator (110) will control theposition of the sample.

X-rays (106) focused by the first X-ray optical device (102) typicallyimpinge on the sample (108), where some X-rays (112, 114) are scatteredor diffracted at various angles by the sample, while other X-rays pass(116) completely through the sample on their original paths without anysignificant deflection. At least some of these X-rays (e.g. at least112) are in turn detected by a detection apparatus (120). Typically, theconfiguration of the X-ray microscope inside the vacuum chamber is suchthat the first X-ray optical device (102) is disposed in between theX-ray port (402) and the sample (108) (e.g. sample position controlledby the sample administrator 110), and the sample (again as controlled bythe sample administrator 110) is substantially in between the firstX-ray optical device (102) and the detection apparatus (112).

This first X-ray optical device (102), as well as optionally the secondX-ray optical device (124) will typically comprise at least one X-raymirror formed from a plurality of curved X-ray reflecting surfaces.Here, X-ray mirror designs such as various Wolter, Kirkpatrick-Baez, orSchwarzschild x-ray mirror designs may be used.

In some embodiments, at least the exposed mirror surfaces of these X-raymirror designs may comprise X-ray resistant, X-ray reflecting materials,such as silicon, boron, silicon triboride, and the like.

As will be discussed shortly, the detection apparatus may contain morethan one portion (e.g. detection region). Usually the detection regionwill comprise at least a first outer region (120) configured to detectboth the location and intensity of various X-rays (112) that have beendiffracted away from the main X-ray beam (116).

Use of three stage vacuum pumps: In operation, to reduce interferencefrom air molecules, which can both absorb and scatter the coherentX-rays, thus reducing microscopy performance, the pressure inside thevacuum chamber (400) will typically be reduced to very low pressures,often on the order of 10⁻⁹ Torr or less, usually by using at least oneexterior vacuum pump system (e.g. 404, 406, 408). This system may be amultiple stage vacuum pump. For example, a three stage vacuum pumpsystem, where the first stage may be a positive displacement pump, thesecond stage may be a sorption pump, and the third stage may be any of acryopump, turbo molecular pump, and/or an ion pump may be used.

The sample administrator (110) will be used to control the position ofthe microscopy sample(s) (108). In some embodiments, such as when thesample administrator (110) holds the sample (108) on the end of a pinlike structure, the sample may be introduced to the vacuum chamber (400)before the pressure is reduced. In other configurations, such as whenthe sample administrator is used to inject or place the sample into thevacuum chamber at a defined location, the sample may be introduced intothe vacuum chamber after the vacuum has been reduced.

Various methods may be used to inject or place the sample. The samplemay be placed into position at the end of a simple pin-like holder, ormore complex robotic sample handing device. Alternatively the sample maybe injected as a stream of small moving particles, as per the methods ofGaffney, Science 316, 1444 (2007).

The X-rays used in this microscopy method are very energetic, and canheat X-ray optical elements (e.g. mirror systems 102, zone plates 124,etc.) to high temperatures. This heating is undesirable, both from thestandpoint of preserving the structure of the X-ray optical elements(102, 124), as well as in causing undesired frequency wavelength shiftsin the X-rays interacting with the X-ray optical elements, thus reducingthe resolution of the microscope. To reduce these effects, thusincreasing both the lifetime and the performance of the various X-rayoptical elements, it will often be useful to chill at least some of thevarious X-ray optical elements (102, 124) to low temperatures,preferably extremely low temperatures such as 20 degrees Kelvin or less.

In at least one mode of operation of the X-ray microscope, the systemwill use the X-ray port (402) to provide a very short burst of coherentX-rays (100), as well as use the first X-ray optical device (102) tofocus and/or collimate this burst of coherent X-rays onto the microscopysample (108) (position determined by the sample administrator 110). Thesystem will then use at least the first portion of the detectionapparatus (120, 120 a) to detect a first signal from the sample. Thisfirst signal will be at least some of the spatially separated X-rays(112, 112 a, 112 b) that have been diffracted at various angles by themicroscopy sample (108).

For at least hard X-ray lasers, very short bursts of illumination arepreferable. This is because the X-ray burst will usually completelydestroy the sample. However if the burst duration is kept extremelyshort, the various atomic components of the sample, although essentiallyexploding outward, will not have enough time to move significantly overthe duration of the burst. Here extremely short bursts of coherentX-rays, often between about 10³¹ ¹³ seconds (100 femtoseconds) and 10⁻¹⁷seconds (10 attoseconds), or even shorter are often preferred. Becausesofter X-rays are much less destructive, however, longer duration burstsmay be used when soft X-ray lasers are used.

At least the first signal (data from the detectors 128) may then be usedto determine the structure of the sample by any means desired. Howeverin a preferred embodiment, the system will use at least one computerprocessor (132) to collect at least first signal data (128) pertainingto the location and intensity of these X-rays (112 a, 112 b), andreconstruct a likely representation of a structure of the microscopysample at approximately a nanometer level of resolution (136). Here,“nanometer level” is typically between about 10 to 0.1 nanometers ofresolution, or more broadly between about 20 to 0.01 nanometers ofresolution.

Thus returning to FIGS. 1 and 4, and to reiterate, coherent X-rays areobtained from an X-ray port (see FIG. 4 402) and are focused andcollimated by a first X-ray optical device (X-ray mirror) (102) andoften an optional pinhole collimator (104). This pulse of focusedcoherent X-rays (106) is then used to illuminate one or more microscopysamples (108) that have been positioned with a sample administrator(110). The pinhole collimator is generally composed of an X-ray opaquematerial with a smaller pinhole. The X-ray opaque material blocksoff-angle X-rays, and the pinhole generally only allows the collimatedor focused X-rays to reach the sample (108).

Put alternatively, the at least one X-ray mirror (102) may be configuredto work in conjunction with a pinhole collimator (104) to concentratecollimated coherent X-rays (106) onto the microscopy sample (108) on orpositioned by the sample administrator (110).

As previously discussed, the sample administrator (110) can be anythingfrom a pin used to place the sample (108) in the correct position, or itmay be a more complex device, such as a sample injection port configuredto dispense a plurality of small droplets of particles of sample intothe vacuum chamber at the correct position and correct times. When thesample administrator is an injection port, the microscope may beconfigured to obtain data from a plurality of similar type samples,(e.g. a plurality of virus particles), and to use this data from aplurality of samples to deduce an average structure of the plurality ofsamples.

As previously discussed, some of the coherent X-rays are diffracted(112) or scattered (114), and the X-rays diffracted at greater anglesconvey information regarding the smaller scale details of the sample.Other coherent X-rays (114) are diffracted or scattered at smallerangles, while many X-rays pass completely through the sample (116). Thusthe smaller angle X-rays (114) tend to be more useful for obtaininglower resolution imaging data (e.g. will report on the larger structuralaspects of the sample), and at the same time, tend to be less useful fordiffraction structure reconstruction purposes because of interferencefrom the main X-ray beam (116).

As previously discussed, in a preferred embodiment of the invention, amore complex set of X-ray optical elements and detectors may be used. Inthis preferred embodiment, the detection apparatus may comprise at leasta first outer portion (120, 120 a) and a second inner portion (126 or126 a or 126 b). This preferred embodiment will also comprise at leastone additional second X-ray optical device (124, or 124 a or 124 b)configured to focus X rays diffracted or scattered from the sample (108)at smaller angles. This second X-ray optical device (124, 124 a, 124 b)will also reside inside vacuum chamber (400). It (124) focuses X-raysobtained from microscopy sample (108) onto a second inner portion ofsaid detection apparatus (126, or 126 a, or 126 b), thereby producing asecond signal from the second X-ray optical device.

Due to the X-ray focusing characteristics of the second X-ray opticaldevice, this second signal is typically a magnified (but lowerresolution) image of the sample (108), as is shown in (134). In thispreferred embodiment, the at least one computer processor (132) willtypically be further configured to use both the first signal (128) fromthe first outer portion of the detection device (120) as well as asecond signal (130) from a second inner portion of the optical detectiondevice (126) to reconstruct a likely representation of a structure ofsaid microscopy sample at a nanometer level of resolution.

The X-rays diffracted at greater angles (112) by sample (108) can bedetected by a first outer portion of an X-ray detection apparatus (120)without need of further X-ray focusing systems. However to make betteruse the smaller angle scattered or diffracted X-rays (114), a secondX-ray optical device may be used. Although this second X-ray opticaldevice may also be an X-ray mirror device, in a preferred embodiment,one or more nanometer scale X-ray zone plates (124) that focus leastsome of the narrower angle X-rays (114) onto a second inner portion ofan X-ray detection apparatus (124) are preferred. These nanometer scaleX-ray zone plates may be fabricated according to the methods ofSakdinawat and Chang, US patent publication 2015/0376798, the entirecontents of which are incorporated herein by reference.

Briefly, these fabrication methods work by forming a metal pattern on asubstrate surface, such as a silicon surface by forming holeconcentration balancing structures onto the substrate proximal to thispattern. Then directionality controlled features are etched from thesubstrate surface into the substrate by metal-assisted chemical etching.The concentration of balancing structures is used to control thedirection of these features. Alternative fabrication methods may also beused.

Put alternatively, in the example configuration shown in FIG. 1, thedetection apparatus comprises a first outer portion (120) comprising alarger multi-pixel solid state X-ray detector configured to capture anddetect most of the greater angle diffracted X-rays. Here this X-raydetector (120) (and often 126 as well) may comprise a plurality ofspatially separated, solid state, detector elements.

In a preferred embodiment, this first outer portion of the detector(120) further comprises a central hole (122) or other type openingthrough which the narrower angle X-rays (114) and main X-ray beam (116)can pass. In this embodiment, on or near the central hole (122) (such ason the other side of the hole) is a second X-ray optical device, such asa nanometer scale zone plate device (124), as well as a second innerportion of the detection apparatus, (126). This second inner portion(126) can either also be a multi-pixel solid state X-ray detector aswell (possibly with different characteristics), or alternatively can bea different type of X-ray detector device.

To illustrate the concepts in more detail, remember that as previouslydiscussed, data (128) from both the first portion of the detectionapparatus (120) and data (130) from the second portion of the apparatus(124) are sent to one or more computer processor(s) (132), which can usethis data to reconstruct a representation of the structure of themicroscopy sample.

In FIG. 1, as an example, assume that the microscopy sample (108) isthat of an adenovirus, such as a type 5 adenovirus that, at a lowerlevel of resolution (134), can be seen to have an overall icosahedralstructure. At a higher level of resolution (136), as we go towards thenanometer level of resolution, we see that the adenovirus has an outercoat composed of hundreds of adenovirus capsomers, which in turn arecomposed of multimeric protomer proteins, each with a characteristicstructure of amino acids arranged in polypeptide chains.

From an X-ray laser microscopy perspective, the higher resolution (e.g.nanometer level) aspects of the adenovirus structure (128, 136) willconvey information pertaining to the internal structure of the variousprotomers, how these protomers in turn assemble into the capsomers, andthe relative spacing of the capsomers. By contrast, the lower resolutionaspects of the adenovirus structure will tend to convey information asto the overall icosahedral shape of the virus, and how the variouscapsomers assemble on a larger scale (130, 134). By using both types ofinformation (130, 134) and (128, 136), the computer processor canproduce a more accurate representation of the adenovirus sample (138)that incorporates both the detailed nanometer scale informationpertaining to capsomer structure, as well as showing the overallicosahedral structure of the adenovirus as well.

FIG. 2A shows a detail of some of the various X-ray laser microscopycomponents previously shown in FIG. 1.

FIG. 2B, shows a drawing of the first outer portion of the X-raydetection apparatus (120) as seen from an orthogonal angle (120 a). Herevarious rays of X-ray light (112 a, 112 b) diffracted from the sample(108) hit the detection apparatus different locations and intensities,creating different patterns of diffraction spots (112 a, 112 b).

FIG. 2C shows a drawing of the second X-ray optical device (e.g. phasezone plate) (124) as seen from an orthogonal angle (124 a), aspreviously discussed. In some embodiments, this phase zone plate can beproduced by high resolution nanofabrication methods. Here an examplefabrication pattern showing various concentric patterns of etched ringsis shown. Although in a preferred embodiment, the first X-ray opticaldevice (102) is an X-ray mirror device, the inventors do not intend toexclude the possibility that the first X-ray optical device may also beone or more phase zone plates also produced by high resolutionnanofabrication methods.

Note that as shown in FIG. 2C, the central portion (125) of the phasezone plate (124 a) may be left solid (e.g. contain no rings, be leftunetched) so as to protect the second inner portion of the detectionapparatus (126) from the damaging effects of the undeflected X-rays(116).

Put alternatively, as previously discussed, the second X-ray opticaldevice (124), as seen from this different angle (124 a), is, in apreferred embodiment, at least one phase zone plate comprising aplurality of nanometer scale phase shifting zone structures configuredto focus or collimate X-rays. In other embodiments, the first X-rayoptical device (102) may be a similar type device.

In other embodiments, the at least one phase zone plate (124) cancomprise one or more multi-layer Laue lenses, such as the lens structuredescribed by H. C. Kang et al., “Nanometer Linear Focusing of Hard XRays by a Multilayer Laue Lens” Phys. Rev. Letts. 96, 127401 (2006).Such multi-layer Laue lenses may, in some embodiments, may also be usedto supplement or replace the first lens (102) or pinhole collimator(104) as well.

FIG. 2D shows a drawing of the second inner portion of the X-raydetection apparatus (126) as seen from an orthogonal angle (126 a). Inthis example, the second X-ray optical device has used some of thenarrow angle X-rays (114) that have been focused (114 a) by the secondX-ray optical device (124), thus forming a magnified image (134 a) ofthe microscopy sample (108).

As previously discussed, the various X-ray optical devices receive muchenergy from the X-ray beams, and thus are at risk for overheating. Thusin some embodiments, the at least one phase zone plate (124, 124 a) maybe further configured with at least one hollow channel where a cryogenicfluid, such as liquid helium, may circulate. This is shown in moredetail in FIG. 3C.

FIG. 3A again shows a detail of some of the various X-ray lasermicroscopy components previously shown in FIG. 1 and FIG. 2A.

FIG. 3B shows that in some embodiments, the first X-ray optical device(102) (e.g. the X-ray mirror device) may be cooled by running a lowtemperature fluid, such as liquid helium, through various hollowchannels (300) in the first X-ray optical device.

FIG. 3C shows that in some embodiments, the second X-ray optical device(124) (e.g. the X-ray phase zone plate) may also be cooled by running alow temperature fluid, such as liquid helium, through various hollowchannels (302) in the second X-ray optical device.

In some embodiments, it may also be useful to cool the entire interiorof the vacuum chamber (400) by running either regular fluids (such aswater or refrigerant fluids such as tetrafluoroethane and/or Freonfamily fluids) or cryogenic fluids (such as liquid nitrogen or evenliquid helium) though various interior channels (410) in the vacuumchamber walls. This is shown in FIG. 4.

FIG. 4 shows some additional components used in the improved X-ray lasermicroscopy method. In a preferred embodiment, components (102) to 126),previously shown in FIG. 1, may be placed inside of a vacuum chamber(400). The coherent X-rays (100) will enter into the vacuum chamber(400) through an X-ray port (402), typically comprised of an X-raytransparent material.

In a preferred embodiment, the vacuum chamber will maintain an ultra-lowvacuum inside the chamber, such as a pressure of 10⁻⁹ Torr or less, andmay optionally employ a sophisticated multiple stage vacuum pump system,such as the previously discussed three stage vacuum pump system (404),(406), (408), to do this.

FIG. 5A shows an alternative embodiment where the second X-ray opticaldevice (phase zone plate) (124) and the second inner portion of thedetection apparatus (126) are disposed in a first alternativeconfiguration with respect to the first outer portion of the detectionapparatus (120) and optional hole (122). Here the second X-ray opticaldevice (124 a) is placed in front of the first outer portion of thedetection apparatus (120), but the second inner portion of the detectionapparatus (126 a) is still placed behind hole (122).

FIG. 5B shows a different alternative embodiment where the second X-rayoptical device (phase zone plate) (124) and the second inner portion ofthe optical detection apparatus (126) are disposed in a secondalternative configuration with respect to the first outer portion of thedetection apparatus (120) and optional hole (122). Here the second X-rayoptical device (124 b) is placed in front of the first outer portion ofthe detection apparatus (120 a), and the second inner portion of thedetection apparatus (126 b) is in the same plane as the first outerportion of the detection apparatus (120). In this embodiment, there isno hole (122).

Other embodiments:

In some embodiments, as shown in FIGS. 1-5B, the first X-ray opticaldevice (102) (e.g. the at least one X-ray mirror) may be entirelyexternal to the pinhole opening in the pinhole collimator (104). Howeverother embodiments are possible. In an alternative embodiment, however,the at least one X-ray mirror may be disposed in, or comprise, thepinhole aperture region (hole) of the pinhole collimator (104).

In some alternative embodiments, both the first X-ray optical device(102) and the second X-ray optical device (124) can be either two phasezone plates (e.g. both configured as per 124 a) or two mirror/collimatordevices (e.g. both configured as per 102/104). In other alternativeembodiments, the first X-ray optical device (102) may be a phase zoneplate, and the second X-ray optical device may be a mirror/collimatordevice (e.g. configured as per 102/104).

In some alternative embodiments, any or all surfaces that receive X-rayphotons (radiation) may be coated with suitable X-ray resistantmaterial, such as silicon, boron, silicon triboride, or other X-rayresistant material.

Software methods: In some embodiments, it may be useful to configure theat least one computer processor (132) to use various machine learningmethods or neural network methods (e.g. deep neural networks, deeplearning methods), to analyze both the X-ray diffraction data (128, 136)as well as X-ray imaging data (130, 134) and deduce an overall compositestructure (138) of the sample (108). This overall structure will thuscombine both sets of data (128, 130) to create a most likely structure(138) that is consistent with the combined data sources. Here, softwarelibraries such as Caffe (developed by Berkeley Vision and Learningcenter), TensorFlow (developed by Google), and the like may be used.

Alternative sample handling devices and methods

FIG. 6 shows a first alternative method of sample handling.

In this first alternative method, the sample administrator (600) cancomprise a revolving multiple container sample holder (602) disposedeither internal or external the vacuum chamber (400) (FIG. 6 shows anexternal configuration). Here the term container means that that thereare individual chambers or spaces in the sample holder that can hold agiven microscopy sample for at least a short duration of time. Althoughthis container will usually have at least some walls, not all walls ofthe container need be sealed. Indeed in some embodiments, the containermay be, for example, a cylinder with one or more open sides (e.g. topand bottom of the cylinder) open to facilitate sample entry,manipulation, and exit. In some cases, entry and exit of the sample intothe container may be controlled by various valves, actuators, and thelike.

In a preferred embodiment, each individual (separate) container (604) ofthis multiple chamber sample holder (602) can be configured to hold aseparate microscopy sample (108), such as a different molecule. Thisseparate microscopy sample can be either a different type of microscopysample (e.g. different types of molecules), or separate versions (e.g.multiple versions) of the same type of sample (e.g. multiple specimensof the same type of molecule).

This first alternative method will typically also use a rotationmechanism (not shown) to control the disposition of the various samples.This rotation mechanism can comprise one or more actuators, oftenelectronically controlled actuators such as motors, solenoids, valvesand the like, and often will be processor controlled to administervarious microscopy samples according to a scheme set up by themicroscope's user. The rotation mechanism controls which sample will beanalyzed at any given time, and here the operation of the rotationmechanism will be termed a “rotation operation”.

In this embodiment, rotation operations on the revolving multiplecontainer sample holder (602) in turn cause any one of the individualcontainers (604) to align with a common sample injection port (606). Ina preferred embodiment, this alignment will be done in such a way as toavoid cross-contamination between different types of samples. This canbe done by small gaps between the containers (604) and the common port(606), use of cleaning mechanisms between samples, and the like.

As a result, possibly facilitated by an injection mechanism (e.g. adevice 610 to inject the given microscopy sample into proper portion ofthe vacuum chamber), the separate microscopy samples (108) from theseparate individual containers (604) are injected into the proper regionof the vacuum chamber (400) (e.g. into the region where the first X-rayoptical device (102) can focus and/or collimate the burst of coherentX-rays onto the microscopy sample (108)), often by way of the commonsample injection port (606).

FIG. 7 shows a second alternative method of sample handling.

In this second alternative method, the “sample administrator” cancomprise a revolving multiple-sample holder (700), often disposedinternal to the vacuum chamber (400). Here separate (again this can beeither different types of samples, or multiple versions of the same typeof sample) microscopy samples (exemplified by 108 a) are disposed ondifferent locations of this revolving multiple-sample holder (700). Themotion of this sample holder can also be controlled by variouselectromechanical actuators, such as motors, solenoids and the like,often also under processor control. These motions will again be termed“rotation operations”.

In this second alternative method, rotation operations on the revolvingmultiple-sample holder (700) cause any one of the separate microscopysamples to be properly positioned for microscopy. Here, again, this willusually be in a position where the first X-ray optical device (102) canfocus and/or collimate a burst of coherent X-rays onto any one of thedifferent microscopy samples (108 a), as selected by the user of themicroscope.

In some embodiments, it may be useful to control the dimensions of therevolving multiple-sample holder and/or to incorporate X-ray shieldingso that samples other than (108 a) that are not being observed areprotected from damage while, for example, sample (108 a) is beinganalyzed.

FIG. 8 shows a third alternative method of sample handling.

This third alternative method of sample handling can be viewed as beingan extension of the second alternative method discussed above. In thisthird alternative method, the revolving multiple sample holder (700 a)(compare with 700 in FIG. 7) further comprises any of a second axis ofrotation and/or a second linear position axis (800).

Here any of these second axis of rotation or second linear position(800), as well as rotation (802) operations on the revolving multiplesample holder (700 a), can cause any one of the separate microscopysamples (108 b) to be properly positioned (e.g. so that the first X-rayoptical device 102 can focus and/or collimate the burst of coherentX-rays onto any one of the different microscopy samples 108 b). AlthoughFIG. 8 (802) shows an embodiment where the multiple sample holder is acylinder, other shapes, such as spheres, cubes, may also be used. Thegeneral idea is simply that the sample holder be configured so that itholds a plurality of samples, and that at least two degrees of movementor rotation may be required to properly position a given microscopysample. In some embodiments, additional shielding may be employed (notshown) to help protect at least some of the samples from the harmfuleffect of the X-rays when the samples are not being observed or analyzedby the X-ray microscope.

FIG. 9A shows a front view of a device useful for a fourth alternativemethod of sample handling.

In this fourth alternative method of sample handling, the sampleadministrator can comprise a plurality of injection ports (e.g. 606 a .. . 606 e), often disposed around a perimeter of the vacuum chamber(400). In FIGS. 9A-9C, these various sample ports are shown disposed ina ring (900) that can, for example form part of the vacuum chamber wall,but other configurations may also be used. Here any individual injectionport (e.g. any one of 606 a . . . 606 e) from this plurality ofinjection ports (606 a . . . 606 e) can be configured to handle aseparate microscopy sample (108), and to inject these separatemicroscopy samples into the vacuum chamber (400) at the proper positionfor viewing by the microscope. As before, these separate microscopysamples can be different types of samples, or multiple versions of thesame type of sample.

FIG. 9B shows a side view of the device from FIG. 9A.

FIG. 9C shows the device from FIG. 9A and 9B in the context of themicroscope's vacuum chamber.

FIG. 10 shows a fifth alternative method of using the microscope toanalyze a plurality of samples. Here the sample administrator cancomprise a plurality of sample holders (108 c, 108 d, 108 e, 108 f, 108g, and 108 h) disposed inside the vacuum chamber (400). Each sampleholder will typically be configured to hold a separate microscopysample. This method uses a robotic sample holder and movement apparatus(1000) (e.g. a robotic hand or arm configured to capture, move, and thenrelease the various sample holders 108 c . . . 108 h).

This robotic sample holder and movement apparatus (1000) is often alsocontrolled by various electromagnetic actuators and optional processorcontrol. The movement apparatus can be used to move any one of thevarious plurality of sample holders (e.g. 108 c) from a storage positioninto a proper position for microscopic analysis. As before, this properposition for microscopic analysis will typically be where the firstX-ray optical device (102) can focus and/or collimate the burst ofcoherent X-rays onto any one of the different microscopy samples.

As before, in some embodiments, it may be useful to control thedimensions and/or location of the storage position sample holder and/orto incorporate X-ray shielding so that samples such as (108 d . . . 108h), that are not being observed, are protected from damage while, forexample, sample (108 c) is being analyzed.

1. An X-ray laser microscopy method, said method comprising: obtainingan X-ray port capable of supplying a coherent source of X-rayillumination; obtaining a first X-ray optical device configured to focusand/or collimate X-rays; obtaining a sample administrator, detectionapparatus, and vacuum chamber, wherein said X-ray port, first X-rayoptical device, and at least a portion of said sample administrator aredisposed inside said vacuum chamber so that said first X-ray opticaldevice is in between said X-ray port and at least one microscopy sampleapplied by said sample administrator, and said at least one microscopysample applied by said sample administrator is substantially in betweensaid first X-ray optical device and said detection apparatus; whereinsaid detection apparatus comprises at least a first outer portion;reducing pressure in said vacuum chamber to a pressure of 10⁻⁹ Torr orless and using said sample administrator to control a position of a saidat least one microscopy sample; chilling said first X-ray optical deviceto temperatures of 20 degrees Kelvin or less; using said X-ray port toprovide a burst of coherent X-rays, and using said first X-ray opticaldevice to focus and/or collimate said burst of coherent X-rays onto saidat least one microscopy sample positioned by said sample administrator;using at least a first portion of said detection apparatus to detect afirst signal comprising spatially separated X-rays that have beendiffracted at various angles from said at least one microscopy sample;and using at least one computer processor and said first signal toreconstruct a likely representation of a structure of said at least onemicroscopy sample at a nanometer level of resolution; wherein saidvacuum chamber comprises walls configured with interior channels, andfurther cooling said vacuum chamber by circulating a fluid through saidinterior channels.
 2. The method of claim 1, wherein said detectionapparatus comprises at least said first outer portion and a second innerportion, further obtaining a second X-ray optical device configured tofocus X rays, disposing said second X-ray optical device inside saidvacuum chamber, and using said second X-ray optical device to focusX-rays obtained from said at least one microscopy sample onto a secondinner portion of said detection apparatus, thereby producing a secondsignal from said second X-ray optical device; and wherein said at leastone computer processor is further configured to use both said firstsignal and said second signal to reconstruct a likely representation ofa structure of said at least one microscopy sample at a nanometer levelof resolution.
 3. The method of claim 2, wherein any of said first orsecond X-ray optical devices comprises at least one phase zone platecomprising a Multilayer Laue Lens or other plurality of nanometer scalephase shifting zones structures configured to focus or collimate X-rays.4. The method of claim 3, wherein at least one said one phase zone plateis further configured with at least one hollow channel, furthercirculating liquid helium through said at least one hollow channel. 5.The method of claim 2, wherein at least said first X-ray optical devicecomprises at least one X-ray mirror formed from a plurality of curvedX-ray reflecting surfaces, said x-ray mirror comprising any of a Wolter,Kirkpatrick-Baez, or Schwarzschild x-ray mirror design.
 6. The method ofclaim 5, wherein said at least one X-ray mirror is configured to work inconjunction with a pinhole collimator to concentrate collimated coherentX-rays onto said at least one microscopy sample applied by said sampleadministrator.
 7. The method of claim 6, wherein said at least one X-raymirror is entirely external to said pinhole collimator, or wherein atleast one X-ray mirror of said at least one X-ray mirror is disposed ina pinhole aperture region (hole) of said pinhole collimator.
 8. Themethod of claim 6, wherein said at least one X-ray mirror or at leastone phase zone plate comprises any of silicon, boron, and silicontriboride.
 9. The method of claim 1, further using a three stage vacuumpump to reduce the pressure in said vacuum chamber to a pressure of10⁻¹³ Torr or less.
 10. The method of claim 1, wherein said burst ofcoherent X-rays has a time duration between 10⁻¹³ seconds (100femtoseconds) and 10⁻¹⁷ seconds (10 attoseconds).
 11. The method ofclaim 1, wherein said at least a first outer portion of said detectionapparatus comprises a plurality of spatially separated, solid state,detector elements.
 12. The method of claim 1, wherein said coherentsource of X-ray illumination is provided by any of a free-electronlaser, capillary plasma-discharge laser, Solid-slab target laser,optical field excited plasma laser, or high-harmonic generation laser.13. The method of claim 1, wherein said sample administrator comprises arevolving multiple container sample holder disposed external to saidvacuum chamber; wherein each separate container of said multiplecontainer sample holder is configured to hold a separate microscopysample; wherein rotation operations on said revolving multiple containersample holder cause any one of said separate containers to align with acommon sample injection port; and wherein separate microscopy samplesfrom different said separate containers are injected into said vacuumchamber through said common sample injection port.
 14. The method ofclaim 1, wherein said sample administrator comprises a revolvingmultiple-sample holder disposed internal to said vacuum chamber; whereinseparate microscopy samples are disposed on different locations of saidrevolving multiple-sample holder; wherein rotation operations on saidrevolving multiple-sample holder cause any one of said separatemicroscopy samples to be positioned so that said first X-ray opticaldevice can focus and/or collimate said burst of coherent X-rays ontosaid any one of said separate microscopy samples.
 15. The method ofclaim 14, wherein said revolving multiple-sample holder furthercomprises either a second axis of rotation or a second linear positionaxis; wherein any of second axis of rotation or second linear positionand said rotation operations on said revolving multiple-sample holdercause any one of said separate microscopy samples to be positioned sothat said first X-ray optical device can focus and/or collimate saidburst of coherent X-rays onto said any one of said separate microscopysamples.
 16. The method of claim 1, wherein said sample administratorcomprises a plurality of injection ports disposed around a perimeter ofsaid vacuum chamber; wherein each individual injection port of saidplurality of injection ports is configured to handle a separatemicroscopy sample, and to inject said separate microscopy samples intosaid vacuum chamber.
 17. The method of claim 1, wherein said sampleadministrator comprises a plurality of sample holders disposed insidesaid vacuum chamber, each said sample holder configured to hold aseparate microscopy sample; further using a robotic sample holdermovement apparatus to move any one of said plurality of said sampleholders so as to cause any one of said separate microscopy samples to bepositioned so that said first X-ray optical device to focus and/orcollimate said burst of coherent X-rays onto said any one of saidseparate microscopy samples.
 18. An X-ray laser microscopy method, saidmethod comprising: obtaining an X-ray port capable of supplying acoherent source of X-ray illumination; obtaining a first X-ray opticaldevice configured to focus and/or collimate X-rays; obtaining a sampleadministrator, detection apparatus, and vacuum chamber, wherein saidX-ray port, first X-ray optical device, and at least a portion of saidsample administrator are disposed inside said vacuum chamber so thatsaid first X-ray optical device is in between said X-ray port and atleast one microscopy sample applied by said sample administrator, andsaid at least one microscopy sample applied by said sample administratoris substantially in between said first X-ray optical device and saiddetection apparatus; wherein said detection apparatus comprises at leasta first outer portion; reducing pressure in said vacuum chamber to apressure of 10⁻⁹ Torr or less and using said sample administrator tocontrol a position of a said at least one microscopy sample; chillingsaid first X-ray optical device to temperatures of 20 degrees Kelvin orless; using said X-ray port to provide a burst of coherent X-rays, andusing said first X-ray optical device to focus and/or collimate saidburst of coherent X-rays onto said at least one microscopy samplepositioned by said sample administrator; using at least a first portionof said detection apparatus to detect a first signal comprisingspatially separated X-rays that have been diffracted at various anglesfrom said at least one microscopy sample; using at least one computerprocessor and said first signal to reconstruct a likely representationof a structure of said at least one microscopy sample at a nanometerlevel of resolution; wherein said vacuum chamber comprises wallsconfigured with interior channels, and further cooling said vacuumchamber by circulating a fluid through said interior channels; andwherein said burst of coherent X-rays has a time duration between 10⁻¹³seconds (100 femtoseconds) and 10⁻¹⁷ seconds (10 attoseconds).
 19. Themethod of claim 18, wherein said sample administrator comprises any of:a) a revolving multiple container sample holder disposed external tosaid vacuum chamber; wherein each separate container of said multiplecontainer sample holder is configured to hold a separate microscopysample; wherein rotation operations on said revolving multiple containersample holder cause any one of said separate containers to align with acommon sample injection port; and wherein different microscopy samplesfrom different said separate containers are injected into said vacuumchamber through said common sample injection port; b) a revolvingmultiple-sample holder disposed internal to said vacuum chamber; whereinseparate microscopy samples are disposed on different locations of saidrevolving multiple-sample holder; wherein rotation operations on saidrevolving multiple-sample holder cause any one of said separatemicroscopy samples to be positioned so that said first X-ray opticaldevice can focus and/or collimate said burst of coherent X-rays ontosaid any one of said separate microscopy samples; said revolvingmultiple-sample holder further comprises either a second axis ofrotation or a second linear position axis; wherein any of second axis ofrotation or second linear position and said rotation operations on saidrevolving multiple-sample holder cause any one of said separatemicroscopy samples to be positioned so that said first X-ray opticaldevice can focus and/or collimate said burst of coherent X-rays ontosaid any one of said separate microscopy samples; d) said sampleadministrator comprises a plurality of injection ports disposed around aperimeter of said vacuum chamber; wherein each individual injection portof said plurality of injection ports is configured to handle a separatemicroscopy sample, and to inject said separate microscopy samples intosaid vacuum chamber; e) said sample administrator comprises a pluralityof sample holders disposed inside said vacuum chamber, each said sampleholder configured to hold a separate microscopy sample; further using arobotic sample holder movement apparatus to move any one of saidplurality of said sample holders so as to cause any one of said separatemicroscopy samples to be positioned so that said first X-ray opticaldevice can focus and/or collimate said burst of coherent X-rays ontosaid any one of said separate microscopy samples.
 20. The method ofclaim 18, wherein said detection apparatus comprises at least said firstouter portion and a second inner portion, further obtaining a secondX-ray optical device configured to focus X rays, disposing said secondX-ray optical device inside said vacuum chamber, and using said secondX-ray optical device to focus X-rays obtained from said at least onemicroscopy sample onto a second inner portion of said detectionapparatus, thereby producing a second signal from said second X-rayoptical device; and wherein said at least one computer processor isfurther configured to use both said first signal and said second signalto reconstruct a likely representation of a structure of said at leastone microscopy sample at a nanometer level of resolution; and whereinany of said first or second X-ray optical devices comprises at least onephase zone plate comprising a Multilayer Laue Lens or other plurality ofnanometer scale phase shifting zones structures configured to focus orcollimate X-rays.